Optoelectronic device and method of fabricating the same

ABSTRACT

A modified isolated polypeptide comprising an amino acid sequence encoding a photocatalytic unit of a photosynthetic organism being capable of covalent attachment to a solid surface and having a photocatalytic activity when attached thereto is disclosed.

RELATED APPLICATIONS

This Application is a Continuation-In-Part (CIP) of PCT PatentApplication No. PCT/IL2006/000241, filed on Feb. 22, 2006, which claimsthe benefit of priority from U.S. patent application Ser. No. 60/654,502filed on Feb. 22, 2005. The contents of the above patent Applicationsare incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to photocatalytic units and, moreparticularly, to solid supports fabricated with same. The presentinvention also relates to an optoelectronic device incorporating thephotocatalytic units and method of fabricating the same.

Nanoscience is the science of small particles of materials and is one ofthe most important research frontiers in modern technology. These smallparticles are of interest from a fundamental point of view since theyenable construction of materials and structures of well-definedproperties. With the ability to precisely control material propertiesarise new opportunities for technological and commercial development,and applications of nanoparticles have been shown or proposed in areasas diverse as micro- and nanoelectronics, nanofluidics, coatings andpaints and biotechnology.

It is well established that future development of microelectronics,magnetic recording devices and chemical sensors will be achieved byincreasing the packing density of device components. Traditionally,microscopic devices have been formed from larger objects, but as theseproducts get smaller, below the micron level, this process becomesincreasingly difficult. It is therefore appreciated that the oppositeapproach is to be employed, essentially, the building of microscopicdevices from a molecular level up, primarily via objects of nanometricdimensions.

Solar cells or photovoltaic cells (PVC) are optoelectronic devices inwhich an incident photonic energy such as sunlight is converted toelectrical power. The use of PVC are as alternative source for renewableenergy gain importance because of the increasing cost of fossil oil, theadverse effect of pollution on the health and on the environment and theprospect of future depletion of the oil reserves. Current technologyuses silicon-based or other types of semiconductor PVCs. PVC are alreadycommercially available and most widely used with an average energyconversion efficiency of 13%. Under research and development arecrystalline and thin layer silicon, GaAs and multi-junction devices someof which can reach up to 30% efficiency. Some of the high efficiency PVCare equipped with concentrating mirrors to reduce the size and thereforethe cost of the PVC. A construction of an optimal PVC cell in which theefficiency per cost ratio is high is yet to be achieved. For thisreason, various types of photo-active materials have been investigatedin addition to Si and GaAs. Several inorganic materials such as CuInSe₂,CdTe/Se, organic and dye synthesized molecules and polymeric films wereinvestigated. Chlorophyll and chlorophyll derivatives were successfullyused as sensitizing dyes in PVC [Radziemska, E. Progress in Energy andCombustion Science 2003, 29, 407-424] over the years. These materialsare also useful for constructing light emitting devices.

Conventionally, these types of solar cells are fabricated by sandwichinga semiconductor p-n junction between a light transmissive electrode andan additional electrode. When a photon enters into the p-n junction,under an appropriate bias voltage, an electron-hole separation takesplace and a photocurrent is generated. Presently known technology usessilicon-based or other types of PVCs. Such devices, however, are costlyand their efficiency is far from being satisfactory. For example,commercially available silicon PVC is known to have an average energyconversion efficiency of 13%. It is expected that crystalline and thinlayer silicon, GaAs and multi-junction devices which are currently underresearch and development, will reach efficiency of 24% for silicon and34% for GaAs. These devices, however, are even more expensive thancommercial silicon PVCs. To reduce cost, compromises are made on thesize and bulkiness of the device. For example, known in the art arephotovoltaic systems which incorporate mirrors to concentrate sunlighton small area of a photovoltaic cell.

Also known, are polymeric and dye-based PVCs. This technology, however,has not yet matured to provide high energy conversion efficiency.Polymeric and dye-based PVCs have reported to provide energy conversionefficiency of 5% or less.

Pigment-protein complexes which are responsible for photosyntheticconversion of light energy to chemical energy may be used as electroniccomponents in a variety of light based devices. Although fabrication ofmolecular circuits is presently beyond the resolution of conventionalpatterning techniques such as electron beam lithography, positioning ofmolecules with sub nanometer precision is routine in nature, and crucialto the operation of biological complexes such as photosyntheticcomplexes.

Green plants, cyanobacteia and photosynthetic bacteria capture andutilize sunlight by means of molecular electronic complexes, reactioncenters that are embedded in their membranes. In oxygenic plants andcyanobacteria, photon capture and conversion of light energy intochemical energy take place in specialized membranes called thylakoids.The thylkoids are located in chloroplast in higher plants or consists offoldings of the cytoplasmic membrane in cyanobacteria. The thylakoids,consisting of stacked membrane disks (called grana) and unstackedmembrane disks (called stroma). The thylakoid membrane contains two keyphotosynthetic components, photosystem I and photosystem II, designatedPS I and PS II, respectively. Photosynthesis requires PSII and PSIworking in sequence, using water as the source of electrons and CO₂ asthe terminal electron acceptor.

PS I is a transmembrane multisubunit protein-chlorophyll complex thatmediates vectorial light-induced electron transfer from plastocyanin orcytochrome C₅₅₃ to ferredoxin. The nano-size dimension, an energy yieldof approximately 58% and the quantum efficiency of almost 1 [K. Brettel,Biochim. Biophys. Acta 1997, 1318 322-373] makes the reaction center apromising unit for applications in molecular nano-electronics.

The crystalline structures of PS I from Synechococus elongatus and fromplant chloroplast were resolved to 2.5 Å at 4.4 Å, respectively [P.Jordan, et al., Nature 2001, 411 909-917; A. Ben Shem, et al., Nature2003, 426 630-635]. In cyanobacteria and plants, the complex consists of12 polypeptides. Some of the polypeptides bind 96 light-harvestingchlorophyll and 22 beta carotenoide molecules. The electron transportchain contains P700, A₀, A₁, F_(X), F_(A) and F_(B) representing achlorophyll a dimmer, a monomeric chlorophyll a, two phylloquinones andthree [4Fe-4S] iron sulfur centers, respectively. The reaction centercore complex is made up of the heterodimeric PsaA and PsaB subunits,containing the primary electron donor, P700, which undergoeslight-induced charge separation and transfers an electron through thesequential carriers A₀, A₁ and F_(X). The final acceptors F_(A) andF_(B) are located on another subunit, PsaC. The redox potential of theprimary donor P700 is +0.43 V and that of the final acceptor F_(B) is−0.53 V producing redox difference of −1.0 V. The charge separationspans about 5 nm of the height of the protein representing the center tocenter distance between the primary donor (P700) and the final acceptor(F_(B)). The protein complex is 9 nm in height and a diameter of 21 nmand 15 for the trimer and the monomer respectively.

It is recognized that in order to incorporate PS I reaction centers intomolecular devices, it is essential to immobilize the PSI reactioncenters onto a substrate without their denaturation.

In earlier works, care was taken to non-covalently attach plant PS I [I.Lee, et al, J. Phys. Chem. B 2000, 104 2439-2443; R. Das, Nano Letters2004, 4 1079-1083] and bacterial reaction centers [C. Nakamura et al.,Applied Biochemistry and Biotechnology 2000, 84-6 401-408; S. A.Trammell, et al., Biosensors & Bioelectronics 2004, 19 1649-1655] tosolid surfaces so as to avoid inactivation of self-assembled monolayers.

Thus, genetic modifications of both a bacterial reaction center [S. A.Trammell, et al., Biosensors & Bioelectronics 2004, 19 1649-1655] and aplant PS I [Das, Nano Letters 2004, 4 1079-1083] by addition of a 6histidine tail allows for non-covalent binding to a polymer coated metalsurface. The histidine attached bacterial reaction center was shown toproduce photocurrent in solution in electrochemical cell. The histidinetagged PS I was shown to be oriented but was not reported to produceeither photocurrent or photopotential. In addition, the histidine taggedPS I as taught by Das supra, required stabilization using peptidesurfactants in order to attach to solid surfaces. Lee et al., [J. Phys.Chem. B 2000] teaches coating a metal surface with organic molecules andadsorbing the PS I non-covalently to the organic layer. In this case,the proteins assumed several orientations. Lee et al., [Biosensors &Bioelectronics, 1996, 11-4, 375-387] teaches platinum precipitation onthe surface of photosynthetic membranes, assuming formation of directelectrical contact with the acceptor side of PS I, because it cancatalyze hydrogen evolution. Additionally, it has been shown thatisolated PS I reaction centers can be platinized since after theplatinization it produced hydrogen in the light.

However, none of these methods teach covalent attachment of functionalPS I reaction centers to a solid surface and certainly not in anoriented manner. PS I reaction centers which are not oriented willcancel each other out, preventing the PS I-immobilized devices to beused in photoelectric devices such as solar batteries or logic gates.

There is thus a widely recognized need for, and it would be highlyadvantageous to synthesize active PS I reaction centers capable ofbinding to a solid surface in an oriented manner, thereby to allowfabrication of optoelectronic device devoid the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amodified isolated polypeptide comprising an amino acid sequence of apolypeptide of a photocatalytic unit of a photosynthetic organism, theamino acid sequence being capable of mediating covalent attachment ofthe photocatalytic unit to a solid surface and maintaining aphotocatalytic activity of the photocatalytic unit when attached to thesolid surface.

According to another aspect of the present invention there is provided aplurality of isolated polypeptides, comprising an amino acid sequence ofa polypeptide of a photocatalytic unit of a photosynthetic organism, theamino acid sequence being capable of mediating covalent attachment ofthe photocatalytic unit to a solid surface and maintaining aphotocatalytic activity of the photocatalytic unit when attached to thesolid surface, being capable of orientating at a substantially similardirection with respect to the solid surface.

According to yet another aspect of the present invention there isprovided an isolated modified photocatalytic unit comprising themodified polypeptide comprising an amino acid sequence of a polypeptideof a photocatalytic unit of a photosynthetic organism, the amino acidsequence being capable of mediating covalent attachment of thephotocatalytic unit to a solid surface and maintaining a photocatalyticactivity of the photocatalytic unit when attached to the solid surface.

According to still another aspect of the present invention there isprovided membrane preparation comprising the modified photocatalyticunit comprising the modified polypeptide comprising an amino acidsequence of a polypeptide of a photocatalytic unit of a photosyntheticorganism, the amino acid sequence being capable of mediating covalentattachment of the photocatalytic unit to a solid surface and maintaininga photocatalytic activity of the photocatalytic unit when attached tothe solid surface.

According to an additional aspect of the present invention there isprovided an isolated polynucleotide encoding the modified polypeptidecomprising an amino acid sequence of a polypeptide of a photocatalyticunit of a photosynthetic organism, the amino acid sequence being capableof mediating covalent attachment of the photocatalytic unit to a solidsurface and maintaining a photocatalytic activity of the photocatalyticunit when attached to the solid surface.

According to yet an additional aspect of the present invention there isprovided a nucleic acid construct comprising the polynucleotide encodingthe modified polypeptide comprising an amino acid sequence of apolypeptide of a photocatalytic unit of a photosynthetic organism, theamino acid sequence being capable of mediating covalent attachment ofthe photocatalytic unit to a solid surface and maintaining aphotocatalytic activity of the photocatalytic unit when attached to thesolid surface.

According to still an additional aspect of the present invention thereis provided a cell comprising the isolated polynucleotide encoding themodified polypeptide comprising an amino acid sequence of a polypeptideof a photocatalytic unit of a photosynthetic organism, the amino acidsequence being capable of mediating covalent attachment of thephotocatalytic unit to a solid surface and maintaining a photocatalyticactivity of the photocatalytic unit when attached to the solid surface.

According to a further aspect of the present invention there is provideda device, comprising a solid surface attached to a plurality of modifiedphotocatalytic units comprising the modified polypeptide comprising anamino acid sequence of a polypeptide of a photocatalytic unit of aphotosynthetic organism, the amino acid sequence being capable ofmediating covalent attachment of the photocatalytic unit to a solidsurface and maintaining a photocatalytic activity of the photocatalyticunit when attached to the solid surface.

According to still a further aspect of the present invention there isprovided an optoelectronic device comprising at least one layer ofphotoactive nanoparticles interposed between a first electrode and asecond electrode, wherein at least one of the first electrode and thesecond electrode is light transmissive.

According to still a further aspect of the present invention there isprovided an optoelectronic array comprising a plurality of theoptoelectronic devices described herein.

According to further features in preferred embodiments of the inventiondescribed below, the second electrode is light transmissive and a workfunction characterizing the second electrode is higher than a workfunction characterizing the first electrode.

According to still further features in the described preferredembodiments the device further comprises a dielectric layer deposited onthe first electrode and having therein a cavity containing the layer(s)of photoactive nanoparticles, wherein the first electrode is exposed ata base of the cavity.

According to still further features in the described preferredembodiments the device further comprises a substrate carrying the firstelectrode and the dielectric layer. The substrate has thereon two ormore electrical contacts, each being in electrical communication withone electrode.

According to still further features in the described preferredembodiments at least a few of the optoelectronic devices share the firstelectrode. According to still further features in the describedpreferred embodiments at least a few of the optoelectronic devices sharethe second electrode.

According to still further features in the described preferredembodiments the plurality of optoelectronic devices comprise: a firstconductive layer having a plurality of electrodes each serving as thefirst electrode; a dielectric layer deposited on the first layer andbeing formed with a plurality of cavities therein, wherein each cavityof the plurality of cavities is positioned above an electrode of thefirst layer and comprises one or more layers of photoactivenanoparticles; and a second conductive layer deposited over the cavitiesand having a plurality of electrodes each serving as the secondelectrode.

According to still a further aspect of the present invention there isprovided a method of fabricating an optoelectronic device, comprisingcovalently attaching photocatalytic units of photosynthetic organisms toat least one first electrode, thereby providing a layer of photoactivenanoparticles on the least one first electrode, and depositing at leastone second electrode on the layer of photoactive nanoparticles.

According to still further features in the described preferredembodiments the method further comprises attaching at least oneadditional layer of photoactive nanoparticles on the layer ofphotoactive nanoparticles.

According to further features in preferred embodiments of the inventiondescribed below, the method further comprises depositing the at leastone first electrode on a substrate.

According to still further features in the described preferredembodiments the method further comprises depositing a dielectric layeron the at least one first electrode and forming a cavity in thedielectric layer so as to expose the at least one first electrode.

According to still further features in the described preferredembodiments the attachment is effected by light induced adsorption.

According to still further features in the described preferredembodiments the deposition of the second electrode comprises sputteringdeposition.

According to still further features in the described preferredembodiments the deposition of the second electrode comprises indirectevaporation.

According to still further features in the described preferredembodiments the photoactive nanoparticles comprise conducting solidsurfaces covalently attached to photocatalytic units of photosyntheticorganisms.

According to further features in preferred embodiments of the inventiondescribed below, the photosynthetic organism is a green plant.

According to still further features in the described preferredembodiments, the photosynthetic organism is a cyanobacteria.

According to still further features in the described preferredembodiments, the photocatalytic unit is PS I.

According to still further features in the described preferredembodiments, the photocatalytic unit is a Synechosystis sp. PCC 6803photocatalytic unit.

According to still further features in the described preferredembodiments, the amino acid sequence of the polypeptide of thephotocatalytic unit comprises at least one substitution mutation.

According to still further features in the described preferredembodiments, the substitution mutation is on an extra-membrane loop ofthe photocatalytic unit.

According to still further features in the described preferredembodiments, the amino acid sequence of the polypeptide is Psa B.

According to still further features in the described preferredembodiments, the amino acid sequence of the polypeptide is Psa C.

According to still further features in the described preferredembodiments, the psa B comprises a substitution mutation in at least oneposition demarked by the coordinates D236C, S247C, D480C, S500C, S600C,Y635C.

According to still further features in the described preferredembodiments, the psa C comprises a substitution mutation in at least oneposition demarked by the coordinates W31C.

According to still further features in the described preferredembodiments, the at least one substitution mutation is cysteine.

According to still further features in the described preferredembodiments, the amino acid sequence is as set forth in SEQ ID NOs: 20,21, 22, 23, 24, 25, 26, 27, 28 and 29.

According to still further features in the described preferredembodiments, the solid surface is a conducting material.

According to still further features in the described preferredembodiments, the conducting material is a transition metal.

According to still further features in the described preferredembodiments, the transition metal is selected from the group consistingof silver, gold, copper, platinum, nickel aluminum and palladium.

According to still further features in the described preferredembodiments, the modified isolated polypeptide does not comprise metalions.

According to still further features in the described preferredembodiments, the modified isolated polypeptide is in a monomeric form ora trimeric form.

According to still further features in the described preferredembodiments, the nucleic acid construct further comprises acis-regulatory element.

According to still further features in the described preferredembodiments, the cis-regulatory element is a promoter.

According to still further features in the described preferredembodiments, the cell is a Synechocystis cell.

According to still further features in the described preferredembodiments, the distance between each of the plurality of modifiedphotocatalytic units is between 15-25 nm.

According to still further features in the described preferredembodiments, the plurality of modified photocatalytic units are orientedwith respect to the solid surface.

According to still further features in the described preferredembodiments, the device serves as a component in a photodiode.

According to still further features in the described preferredembodiments, the device serves as a component in a phototransistor.

According to still further features in the described preferredembodiments, the device serves as a component in a logic gate.

According to still further features in the described preferredembodiments, the device serves as a component in a solar cell.

According to still further features in the described preferredembodiments, the device serves as a component in an optocoupler.

According to still further features in the described preferredembodiments, the plurality of modified photocatalytic units are directlyattached to the solid surface.

According to still further features in the described preferredembodiments, the directly attached is covalently attached.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in colorphotograph. Copies of this patent with color photograph(s) will beprovided by the Patent and Trademark Office upon request and payment ofnecessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-E describe the cysteine mutations in PS I and provide evidencethat the cysteine mutations are on the external surface of PS I. FIGS.1A-C are schemes of the vectors used for induction of mutations in psaBof Synechocystis sp. PCC 6803 by homologous recombination. Plasmid pZBLwas constructed by insertion of a 1.8 kb fragment of the psaB gene, akanamycin resistance conferring gene (Kan^(R)) and the 1.1 kb downstreamflanking region into the pBluescript vector (FIG. 1B). For selection ofa psaB deficient recipient cells a pBLΔB vector was constructed byremoval of 1.3 kb of downstream end of psaB and insertion of aChloramphenicol resistant gene (Cm^(R)) (FIG. 1C). The restriction siteson the genomic DNA are indicated in FIG. 1A. FIG. 1D is a backbonepresentation of the structure of PS I with the proposed mutations in“spacefill”. The arrow shows the direction of light induced chargetransfer. The amino acids in the PsaB subunit that were mutated tocysteines are displayed from left to right as the following: D236C,S247C, D480C, S500C, S600C, Y6354C. The coordinates were taken from PDBfile JBO1 and displayed with the aid of RasMole software. FIG. 1E is aphotograph of an immunoblot of surface-exposed cysteines on isolatedwild type PS I complexes (lane 1) and genetically modified PS Icomplexes (lanes 2-7).

FIGS. 2A-B are images of two-dimensional spatial arrays of oriented PS Ireaction centers on a gold surface. The images were obtained bytapping-mode atomic force microscopy (AFM; 0.3 μm² area). FIG. 2A is animage of annealed 150 nm gold surface on a silicon slide and FIG. 2B isan image of gold substrate that was incubated in a solution containingPS I monomers of mutant D480C polypeptide (SEQ ID NO: 20).

FIGS. 3A-B are two-dimensional spatial and electric potential maps oforiented PS I reaction centers on gold surfaces. Topographic (FIG. 3A)and electric potential (FIG. 3B) images of the same set of PS I reactioncenter trimers from mutant D480C (SEQ ID NO: 20) on a Au—Si surface. Alight-induced PS I negative electrical potentials of PS I is seen inFIG. 3B. The illumination was provided by a He—Ne laser at 632.8 nm, 5mW/cm² where indicated on the image. The negative sign of the potentialshown in FIG. 3B is due to the KPFM feedback circuit and is opposite tothe actual sign of the CPD.

FIGS. 4A-B are two-dimensional spatial and electric potential maps oforiented PSI reaction centers on gold surface. Topographic (FIG. 4A) andelectric potential (FIG. 4B) images of the same set of PS I reactioncenters trimers from mutant D480C on a Au—Si surface. A light-induced PSI negative electrical potentials of PS I is seen in FIG. 4B. Thescanning directions for each raster of the constructed images were fromtop to bottom (from light to dark). The illumination was provided by aHe—Ne laser at 632.8 nm, 5 mW/cm². The negative sign of the potential asshown in FIG. 4B is due to the KPFM feedback circuit and is opposite tothe actual sign of the CPD.

FIGS. 5A-B are three-dimensional topographic and electric potentialimages of oriented PS I reaction centers on gold surface. FIG. 5Aillustrates an electric potential image of PS I reaction centersmonomers on a Au—Si surface. A light-induced PS I negative electricalpotential of PS I is seen on turning the illumination by a He—Ne laserat 632.8 nm, 5 mW/cm² (hv). The scanning directions for each raster ofthe constructed images were from top to bottom (from dark to light).FIG. 5B illustrates a three dimensional topographic presentation of PS Itrimer on Au—Si substrate.

FIGS. 6A-B are spectroscopic measurements of PS I. FIG. 6A illustratesflash induced transient absorption changes of P700 in an isolated PS Imutant (SEQ ID NO: 20). The absorption changes of P700 were monitored at820 nm (ΔA₈₂₀) following a saturating laser flash in D480C mutant PS Icomplexes. FIG. 6B illustrates X-ray absorption spectroscopy of orientedPS I complexes. PS I orientation in self assembled monolayer isdetermined by total reflection measurements of grazing x-rayfluorescence. PS I was attached through formation of sulfide bondbetween unique cysteine and tungsten on tungsten-carbon multilayer oversilicon substrate. Each graph is an average of 60, 42 s scans in theindicated angle to the x-ray beam normal 25 (—), 45 (Δ), 60 (−) and 90(O) degrees.

FIG. 7 is a schematic illustration of an optoelectronic device,according to various exemplary embodiments of the present invention.

FIG. 8 is schematic illustration of a photodiode device, according tovarious exemplary embodiments of the present invention.

FIG. 9 is a schematic illustration of a phototransistor, according tovarious exemplary embodiments of the present invention.

FIG. 10 is a simplified illustration of an optocoupler, according tovarious exemplary embodiments of the present invention.

FIGS. 11 a-b are simplified illustrations of an optoelectronic device,according to various exemplary embodiments of the present invention.

FIG. 12 illustrates an energy-level diagram in the preferred embodimentin which one electrode of the device is made of aluminum and anotherelectrode is made of indium tin oxide.

FIGS. 13 a-b are schematic illustrations of an optoelectronic array,according to various exemplary embodiments of the present invention.

FIG. 14 is a flowchart diagram (FIG. 14) of a method suitable forfabricating an optoelectronic device, according to various exemplaryembodiments of the present invention

FIGS. 15 a-d are schematic process illustrations of various method forfabricating the optoelectronic device, according to various exemplaryembodiments of the present invention.

FIGS. 16 a-d are schematic process illustrations of various method stepsfor fabricating the optoelectronic array, according to various exemplaryembodiments of the present invention.

FIGS. 17 a-c are two-dimensional spatial and electric potential maps oforiented PSI reaction centers on gold surface, in various exemplaryembodiments of the invention. FIGS. 17 a-b are topographic (FIG. 17 a)and electric potential (FIG. 17 b) images of the same set of PS Ireaction center trimers from mutant D480C on an Au—Si surface. Alight-induced PS I negative electrical potentials of PS I is seen inFIG. 17 b. The scanning directions for each raster of the constructedimages were from top to bottom (from light to dark). The illuminationwas provided by a He—Ne laser at 632.8 nm, 5 mW/cm2. The negative signof the potential as shown in the figure is due to the KPFM feedbackcircuit and is opposite to the actual sign of the CPD50. FIG. 17 c showsbinding PS I under illumination.

FIG. 18 a-d are images obtained by atomic force microscopy of platinizedPS I monolayer. FIGS. 18 a and 18 c are topographic image of PS I (FIG.18 a) an platinized (FIG. 18 c). FIGS. 18 b and 18 d show the respectivephase contrast images. The features of the protein are damped by themetal in the phase images.

FIG. 19 shows electrochemical measurements of photocurrent in PS Imonolayer on gold electrode. The working electrode was illuminated (onand off) from a 150 W slide projector through the glass wall of thecell. The medium contained 0.1 M tris-HCl, pH 7.5 and 0.05 mM methylviologene.

FIGS. 20 a-c are images of a prototype optoelectronic array fabricatedaccording to various exemplary embodiments of the present invention.FIG. 20 a shows chemically adsorbed layer of PS I molecules (whitedots); FIG. 20 b is an electron microscopy image of a cavity whichexposes a gold electrode; FIG. 20 c is optical microscopy image of theprototype showing an array of optoelectronic devices sandwiched betweenthe bottom gold electrodes (“source”) and the top indium tin oxideelectrode (“drain”).

FIG. 21 is a schmatic illustration of the prototype optoelectronic arrayof FIGS. 20 a-c.

FIG. 22 is a schematic illustration of set-up used for photoconductivityexperiments performed using the prototype device of FIGS. 20 a-c.

FIG. 23 the photoconductivity I/V data obtained from the experimentillustrated in FIG. 22.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present embodiments comprise a modified photocatalytic unit whichcan be covalently attached to a solid support and maintain activity.Specifically, the present embodiments can be used as electroniccomponents in a variety of different devices, include, withoutlimitation, spatial imaging devices, solar batteries, optical computingand logic gates, optoelectronic switches, photonic A/D converters, thinfilm photovoltaic structures and the like.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description, illustrated in the drawings orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

Photosynthesis is the biological process that converts electromagneticenergy into chemical energy through light and dark reactions. Inoxygenic plants and cyanobacteria, photon capture and conversion oflight energy into chemical energy take place in specialized membranescalled thylakoids. The thylkoids are located in chloroplast in higherplants or consists of foldings of the cytoplasmic membrane incyanobacteria.

PS I is a transmembrane multisubunit protein-chlorophyll complex thatmediates vectorial light-induced electron transfer from plastocyanin orcytochrome C₅₅₃ to ferredoxin. The nano-size dimension, an energy yieldof approximately 58% and the high quantum efficiency makes the reactioncenter a promising unit for applications in molecular nano-electronics.However, in order to incorporate PS I reaction centers into moleculardevices, it is essential to immobilize the PS I reaction centers onto asubstrate without their denaturation. In addition, an orientedattachment of the PS I reaction centers is imperative so that theinduced electrical charges will not cancel each other out.

While reducing the present invention to practice, the present inventorsdiscovered that polypeptides in photocatalytic units may be geneticallymodified such that they comprise functional groups for covalent bindingto a solid surface whilst still retaining activity.

As illustrated in the Examples section which follows, utilizing publiclyavailable structural data on the 3D structure of PS I, the presentinventors mutated amino acids in the Psa B polypeptide and Psa Cpolypeptide of the PS I in the extra membrane loops facing thecytoplasmic side of the bacterial membrane to cysteines in order toensure formation of sulfide bonds (between the PS I unit and a metalsurface). The various mutations were selected near the P700 to secureclose proximity between the reaction center and the gold electrode inorder to facilitate efficient electric junction (Example 1). The presentinventors also showed that by substituting an identical amino acid forcysteine in a plurality of photocatalytic units, the attachment to asolid support will be oriented and the PS I units would form a monolayeron the solid support (Example 2). The precise orientation of thephotocatalytic units on the solid support may be adjusted by selecting aparticular amino acid to be substituted by the cysteine residue.

PS I units modified according to the above were capable of formingoriented monolayers on gold surfaces as detected by atomic forcemicroscopy—FIGS. 2A-B. The mutant PS I units were functionally activefollowing attachment to a gold surface as demonstrated by their abilityto produce a clear light-induced electric potential as measured byKelvin probe force microscopy (KPFM)—FIGS. 3A-B and by their ability totransfer electrons as measured by single turnover spectroscopy [FIGS.6A-B].

Trammel et al., [Biosensors & Bioelectronics 2004, 19 1649-1655] teachesa modification to a bacterial reaction center comprising a 6 histidinetail. In sharp contrast to the present invention, the mutated PS Iscould not covalently bind to a metal surface.

Das [Nano Letters 2004, 4 1079-1083] teaches a modification to a plantreaction center comprising a 6 histidine tail. As well as not covalentlybinding to a metal surface, in sharp contrast to the present invention,the mutated PS Is required stabilization with peptide surfactants inorder to attach to a solid surface.

Lee et al., [J. Phys. Chem. B 2000] teaches coating a metal surface withorganic molecules and adsorbing the PS I non-covalently to the organiclayer. In contrast to the present invention, the PS I proteins assumedseveral orientations and as such were functionally inactive.

Lee et al., [Biosensors & Bioelectronics, 1996, 11-4, 375-387] teachesplatinum precipitation on the surface of photosynthetic membranes,thereby making direct electrical contact with the acceptor side of PS I,where it can catalyze hydrogen evolution. The potential capacity todrive photocurrent through an external circuit was not demonstrated inthese studies. Similarly to Trammel et al and Das, Lee et al does notteach covalent attachment of bacterial reaction center and PS I,respectively to a solid surface. In sharp contrast to Trammell, et al.,and Das, the PS I modified proteins of the present invention need not bemodified to comprise metal ions since they bind to a metal surface byvirtue of the introduced cysteinyl residues located at theextra-membrane loops in PS I.

Thus, according to one aspect of the present invention there is provideda modified isolated polypeptide comprising an amino acid sequence of apolypeptide of a photocatalytic unit of a photosynthetic organism, theamino acid sequence being capable of mediating covalent attachment ofthe photocatalytic unit to a solid surface and maintaining aphotocatalytic activity of the photocatalytic unit when attached to thesolid surface.

As used herein, the phrase “photocatalytic unit” refers to a complex ofat least one polypeptide and other small molecules (e.g. chlorophyll andpigment molecules), which when integrated together work as a functionalunit converting light energy to chemical energy. As mentioned hereinabove, the photocatalytic units of the present invention are present inphotosynthetic organisms (i.e. organisms that convert light energy intochemical energy). Examples of photosynthetic organisms include, but arenot limited to green plants, cyanobacteria, red algae, purple and greenbacteria.

Thus, examples of photocatalytic units which can be used in accordancewith this aspect of the present invention include biologicalphotocatalytic units such as PS I and PS II, bacterial light-sensitiveproteins, bacterial light-sensitive proteins, bacteriorhodopsin,photocatalytic microorganisms, pigments (e.g., proflavine andrhodopsin), organic dyes and algae. Preferably, the photocatalytic unitof the present invention is photosystem I (PS I).

PS I is a protein-chlorophyll complex, present in green plants andcyanobacteria, that is part of the photosynthetic machinery within thethylakoid membrane. It is ellipsoidal in shape and has dimensions ofabout 9 by 15 nanometers.

As used herein the term “about” refers to ±10%.

The PS I complex typically comprises chlorophyll molecules which serveas antennae which absorb photons and transfer the photon energy to P700,where this energy is captured and utilized to drive photochemicalreactions. In addition to the P700 and the antenna chlorophylls, the PSIcomplex contains a number of electron acceptors. An electron releasedfrom P700 is transferred to a terminal acceptor at the reducing end ofPSI through intermediate acceptors, and the electron is transportedacross the thylakoid membrane.

Examples of PS I polypeptides are listed below in Table 1 together withtheir source organisms.

TABLE 1 Source Organism Protein accession number Amphidinium carteraeCAC34545 Juniperus chinensis CAC87929 Cedrus libani CAC87143Spathiphyllum sp. SM328 CAC87924 Persea americana CAC87920 Zamia pumilaCAC87935 Ophioglossum petiolatum CAC87936 Taxus brevifolia CAC87934Afrocarpus gracilior CAC87933 Pinus parviflora CAC87932 Picea spinulosaCAC87931 Phyllocladus trichomanoides CAC87930 Serenoa repens CAC87923Saururus cernuus CAC87922 Platanus racemosa CAC87921 Pachysandraterminalis CAC87919 Nymphaea sp. cv. Paul Harriot CAC87918 Nuphar luteaCAC87917 Nelumbo nucifera CAC87916 Acer palmatum CAD23045 Cupressusarizonica CAC87928 Cryptomeria japonica CAC87927 Abies alba] CAC87926Gnetum gnemon CAC87925 Magnolia grandiflora CAC87915 Liquidambarstyraciflua CAC87914 Lilium brownii CAC87913 Isomeris arborea CAC87912Fagus grandifolia CAC87911 Eupomatia laurina CAC87910 Enkianthuschinensis CAC87909 Coptis laciniata CAC87908 Chloranthus spicatusCAC87907 Calycanthus occidentalis CAC87906 Austrobaileya scandens]CAC87905 Amborella trichopoda CAC87904 Acorus calamus CAC87142

According to a preferred embodiment of this aspect of the presentinvention, the PS I is derived from cyanobacteria and more specificallyfrom Synechosystis sp. PCC 6803.

In cyanobacteria, the PS I complex consists of 12 polypeptides, some ofwhich bind 96 light-harvesting chlorophyll and 22 beta carotenoidmolecules. The electron transport chain contain P700, A₀, A₁, F_(X),F_(A) and F_(B) representing a chlorophyll a dimmer, a monomericchlorophyll a, two phylloquinones and three [4Fe-4S] iron sulfurcenters, respectively. The reaction center core complex is made up ofthe heterodimeric PsaA and PsaB subunits, containing the primaryelectron donor, P700, which undergoes light-induced charge separationand transfers an electron through the sequential carriers A₀, A₁ andF_(X). The final acceptors F_(A) and F_(B) are located on anothersubunit, PsaC.

PS Is derived from cyanobacteria are more structurally stable than thosederived from plant and bacterial reaction centers. This is due to thefact that all chlorophyll molecules and carotenoids are integrated intothe core subunit complexes in cyanobacteria while in plant and otherbacterial reaction centers the antenna chlorophylls are bound tochlorophyll-protein complexes that are attached to the core subunits.Thus, unlike PS Is derived from other organisms such as plants and otherbacteria., those derived from cyanobacteria do not require peptidesurfactants for stabilization [R. Das et al., Nano Letters 2004, 41079-1083] during attachment to a solid surface.

As used herein, the term “isolated” refers to the modifiedphotocatalytic polypeptide that has been at least partially removed fromits natural site of synthesis (e.g., photosynthetic organism).Typically, the photocatalytic polypeptide is not isolated from othermembers of the photocatalytic unit (i.e. chlorophyll and pigment) sothat the photocatalytic unit remains functional. Preferably thepolypeptide is substantially free from other substances (e.g., othercells, proteins, nucleic acids, etc.) that are present in its in-vivolocation.

As mentioned, the photocatalytic unit of this aspect of the presentinvention comprises the modified polypeptide.

The term “polypeptide” as used herein refers to a polypeptide which maybe synthesized by recombinant DNA technology.

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 2 and 3 below list naturally occurring amino acids (Table 2) andnon-conventional or modified amino acids (Table 3) which can be usedwith the present invention.

TABLE 2 Three-Letter One-letter Amino Acid Abbreviation Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 3 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α ethylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylomithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α thylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α ethylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-αthylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucineMleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α ethylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe nbhm N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbchylamino)cyclopropane

As used herein, the phrase “modified polypeptide” refers to apolypeptide comprising a modification as compared to the wild-typepolypeptide. Typically, the modification is an amino acid modification.Any modification to the sequence is envisaged according to this aspectof the present invention so long as the polypeptide is capable ofcovalent attachment to a solid surface and retains a photocatalyticactivity. Examples of modifications include a deletion, an insertion, asubstitution and a biologically active polypeptide fragment thereof.Insertions or deletions are typically in the range of about 1 to 5 aminoacids.

The site of modification is selected according to the suggested 3Dstructure of the photocatalytic unit. Evidence relating to the 3Dstructure of photocatalytic units may be derived from X-raycrystallography studies or using protein modeling software. Thecrystalline structure of PS I from Synechococus elongatus and fromplants chloroplast has been resolved to 2.5 Å at 4.4 Å, respectively [P.Jordan, et al., Nature 2001, 411 909-917; A. Ben Shem, F. Frolow, N.Nelson, Nature 2003, 426 630-635].

The amino acid to be replaced or the site of insertion is typically onthe external surface of the photocatalytic unit (e.g. on an extramembrane loop). Preferably, the amino acids to be replaced or the siteof insertion is in a position which does not cause steric hindrance.Also it is preferred that the mutations are positioned near the P700 ofthe photocatalytic unit to secure close proximity between the reactioncenter and the solid surface in order to facilitate an efficientelectric junction.

According to a preferred embodiment of this aspect of the presentinvention, the modification is a substitution (i.e. replacement)comprising a functional group side chain which is capable of mediatingbinding to the solid surface. Particularly preferred coordinates formutation of PS I from Synechocystis sp. PCC 6803 in PsaB include singlemutations D236C, S247C, D480C, S500C, S600C, and Y635C or doublemutations D236C/Y635C and S247C/Y635C. In PsaC, a particularly preferredsite for a mutation is W31C. In addition, a triple mutation may begenerated in the photocatalytic units (e.g. PsaC//PsaBW31C//D236C/Y635C).

Preferably, the wild type amino acid which is substituted is notessential for the activity of the photocatalytic unit. Guidance indetermining which amino acids are functionally redundant may be found bycomparing the sequence of the photocatalytic unit with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology.

In one embodiment, conservative amino acid substitutions are made at oneor more predicted, non-essential amino acid residues. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined within-the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

In another embodiment, non-conservative amino acid substitutions may bemade since the mutations are preferably designed to enable orientedcovalent attachment of the protein to metal. By selecting mutant cellsthat can grow photoautotrophically, undamaged PS I cells may be ensured.

Preferably, the amino acids at the coordinates described hereinabove arereplaced with an amino acid which is capable of binding to a metalsurface—e.g. amino acids that comprise a thiol group such as cysteine.

In a preferred embodiment of this aspect of the present invention, thesequences of the polypeptides are as set forth in SEQ ID NOs: 20, 21,22, 23, 24, 25, 26, 27, 28 and 29.

Recombinant techniques are preferably used to generate the polypeptidesof the present invention since photocatalytic units typically comprisemore than one polypeptide and other molecules (e.g. pigment moleculesand chlorophyll) integrated into a complex. In addition, thesetechniques are better suited for generation of relatively longpolypeptides (e.g., longer than 20 amino acids) and large amountsthereof. Such recombinant techniques are described by Bitter et al.,(1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

The polypeptides of the present invention may be modified by standardtechniques, such as site-directed mutagenesis (oligonucleotide-mediatedmutagenesis) and PCR-mediated mutagenesis. Thus a polynucleotideencoding a polypeptide of a photocatalytic unit may be mutated.Following mutagenesis the photocatalytic units can be expressed in anappropriate cell system.

Oligonucleotide-mediated mutagenesis is a technique which is well knownin the art as described by Adelman et al., DNA, 2: 183 (1983). Briefly,a polynucleotide encoding a polypeptide of a photocatalytic unit (e.g.PsaB gene) is altered by hybridizing an oligonucleotide encoding thedesired mutation to a polynucleotide template, where the template is thesingle-stranded form of the plasmid containing the unaltered or nativepolynucleotide sequence of the polypeptide of the photocatalytic unit.After hybridization, a DNA polymerase (e.g. Klenow fragment of DNApolymerase I) is used to synthesize an entire second complementarystrand of the template that will thus incorporate the oligonucleotideprimer, and will code for the selected alteration in the photocatalyticunit polynucleotide, thus producing a heteroduplex molecule.

This heteroduplex molecule is then transformed into a suitable hostcell, usually a prokaryote such as E. coli JM101. After the cells aregrown, they may be plated onto agarose plates and screened identify thebacterial colonies that contain the mutated DNA. The mutated region isthen removed and placed in an appropriate vector for protein production,generally an expression vector of the type typically employed fortransformation of an appropriate host.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-strandedpolynucleotide template molecule. The oligonucleotides are readilysynthesized using techniques known in the art such as that described byCrea et al., Proc. Natl. Acad. Sci. USA, 75: 5765 (1978).

The polynucleotide template can only be generated by those vectors thatare either derived from bacteriophage M13 vectors (the commerciallyavailable M13 mp18 and M13 mp19 vectors are suitable), or those vectorsthat contain a single-stranded phage origin of replication as describedby Viera et al. Meth. Enzymol., 153: 3 (1987). Thus, the polynucleotidethat is to be mutated must be inserted into one of these vectors togenerate single-stranded template. Production of the single-strandedtemplate is described in Sections 4.21-4.41 of Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, N.Y. 1989)

Mutants with more than one amino acid to be substituted may also begenerated using site directed mutagenesis.

An exemplary method for mutating PS I according to the teachings of thepresent invention, using site-directed mutagenesis is described inExample 1 herein below.

PCR mutagenesis and cassette mutagenesis are also techniques that aresuitable for modifying polypeptides of photocatalytic units—See Sambrookand Russell (2001, Molecular Cloning, A Laboratory Approach, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.) and Ausubel et al. (2002,Current Protocols in Molecular Biology, John Wiley & Sons, NY).

Suitable hosts for the expression of the mutated photocatalyticsequences include any host that is capable of synthesizing a functionalphotocatalytic unit. Thus the host must be capable of incorporatingpigment, chlorophyll molecules and the like into the unit. Examples ofsuitable hosts include, but are not limited to green plant cellcultures, green plants and photosynthetic bacteria. In a preferredembodiment of this aspect of the present invention, the host isSynechocystis bacteria.

According to a particularly preferred embodiment of the presentinvention, the mutated DNA is cloned by insertion into the host genome.This is particularly suitable when the host cell are photosyntheticbacteria. This method is affected by including in the vector a DNAsequence that is complementary to a sequence found in the photosyntheticgenomic DNA. Transfection of photosynthetic bacteria with this vectorresults in homologous recombination with the genome and insertion of thephotocatalytic poypeptide DNA.

An exemplary method for the transformation by homologous recombinationof Synechocystis sp. PCC 6803 with a mutated psaB gene is described inExample 1 below. Essentially, Wild-type Synechocystis cells,light-activated heterotrophically grown (LAHG: grown in the dark exceptfor 10 minutes of light at photon flux density of 40 micromolm_(—2)s_(—1) every 24 h) on BG-11 plates supplemented with 5 mM glucose,10 mM TES-KOH, pH 8(N-tris[Hydroxymethyl]-methyl-2-aminoethanesulfonate) and thiosulfate (3g/l), were transformed with the plasmid pZBL cloned with the mutatedpsaB gene. A scheme for homologous recombination is depicted in FIG. 1A.The cells were transformed with the resultant plasmids, then selectedand segregated for a few generations on 5-20 mg/ml kanamycin. Selectionof transformants and segregation was performed under kanamycin pressure.

Alternatively a polynucleotide encoding a modified polypeptide of thepresent invention may be ligated into a nucleic acid expression vector,which comprises the polynucleotide sequence under the transcriptionalcontrol of a cis-regulatory sequence (e.g., promoter sequence) suitablefor directing constitutive, tissue specific or inducible transcriptionof the polypeptides of the present invention in the host cells.

The phrase “an isolated polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above). Examples of polynucleotidesequences which may be used according to the teachings of the presentinvention are as set forth in SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37and 38.

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

As mentioned hereinabove, polynucleotide sequences of the presentinvention may be inserted into expression vectors (i.e., a nucleic acidconstruct) to enable expression of the recombinant polypeptide. Theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).Typical cloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for thepolypeptide expressed. For example, when large quantities ofpolypeptides are desired, vectors that direct the expression of highlevels of the protein product, possibly as a fusion with a hydrophobicsignal sequence, which directs the expressed product into the periplasmof the bacteria or the culture medium where the protein product isreadily purified may be desired. Certain fusion protein engineered witha specific cleavage site to aid in recovery of the polypeptide may alsobe desirable. Such vectors adaptable to such manipulation include, butare not limited to, the pET series of E. coli expression vectors[Studier et al., Methods in Enzymol. 185:60-89 (1990)].

In cases where plant expression vectors are used, the expression of thepolypeptide coding sequence can be driven by a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat proteinpromoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] can beused. Alternatively, plant promoters can be used such as, for example,the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680(1984); and Brogli et al., Science 224:838-843 (1984)] or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol.Cell. Biol. 6:559-565 (1986)]. These constructs can be introduced intoplant cells using Ti plasmid, Ri plasmid, plant viral vectors, directDNA transformation, microinjection, electroporation and other techniqueswell known to the skilled artisan. See, for example, Weissbach &Weissbach [Methods for Plant Molecular Biology, Academic Press, NY,Section VII, pp 421-463 (1988)].

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media.

Various methods can be used to introduce the expression vector of thepresent invention into the host cell system. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Transformed cells are cultured under effective conditions, which allowfor the expression of high amounts of recombinant polypeptide. Effectiveculture conditions include, but are not limited to, effective media,bioreactor, temperature, pH and oxygen conditions that permit proteinproduction. An effective medium refers to any medium in which a cell iscultured to produce the recombinant polypeptide of the presentinvention. Such a medium typically includes an aqueous solution havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Following culturing under suitable conditions, the photocatalytic unitsare preferably isolated from the cells. An exemplary method for removingphotocatalytic units from photosynthetic organisms is described inExample 2 of the Examples section hereinbelow. The present inventionalso envisages using any other methods of purification and isolation solong as the photocatalytic unit remains functional. The photocatalyticunits may be isolated as polymers e.g. trimers or as single monomers.The photocatalytic units may be fully isolated or part of a membranepreparation. Methods of preparing membrane extracts are well known inthe art. For example, Qoronfleh et al., [J Biomed Biotechnol. 2003;2003(4): 249-255] teach a method for selective enrichment of membraneproteins by partition phase separation. Various kits are alsocommercially available for the preparation of membrane extracts such asfrom Sigma-Aldrich (ProteoPrep™ Membrane Extraction Kit).

Thus, according to another aspect of the present invention, there isprovided an isolated modified photocatalytic unit comprising themodified polypeptide of the present invention. According to this aspectof the present invention, the term “isolated” refers to photocatalyticunit that has been at least partially removed from its natural site ofsynthesis (e.g., photosynthetic organism). Preferably the photocatalyticunit is substantially free from substances (e.g., other cells, proteins,nucleic acids, etc.) that are present in its in-vivo location.

The activity of the photocatalytic units may be tested followingisolation as described hereinbelow.

Following isolation, the modified photocatalytic units of the presentinvention may be attached to a solid surface by covalent or non-covalentbonding (electrostatic). As used herein the term “covalent bond” refersto the linkage of two atoms by the sharing of two electrons, onecontributed by each of the atoms. Preferably the photocatalytic unit isbonded directly to a solid surface (i.e. does not comprise any linkermolecules nor is it coated with metal ions).

Selection of a solid surface depends on the modification of thephotocatalytic unit. Thus, if the modification comprises a cysteinesubstitution, as exemplified hereinbelow, the solid surface ispreferably a conducting material, such as a transition metal. Examplesof transition metals which may be used according to this aspect of thepresent invention include, but are not limited to silver, gold, copper,platinum, nickel, aluminum and palladium.

The modified photocatalytic unit of the present embodiments can becovalently attached to a solid surface by directly reacting thesubstituting residue with a hydrophilic surface of a solid substrate.For example, in the preferred embodiment is which the substitutingresidue is cysteine, the attachment can be done by incubating themodified photocatalytic unit with gold or other metals surfaces for aperiod sufficient to form a sulfide bond. Other attachment methods arealso contemplated. An exemplary method for covalently attaching acysteine substituted photocatalytic unit is described in Example 2 ofthe Examples section herein below.

According to this aspect of the present invention, the modifiedphotocatalytic unit retains photocatalytic activity following attachmentto a solid surface.

Herein, the phrase “photocatalytic activity” refers to the conversion oflight energy to chemical energy. Preferably, the modified photocatalyticunits retain at least 20%, more preferably at least 30%, more preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, morepreferably at least 90%, e.g., about 100% the activity of the wild typephotocatalytic unit in its in-vivo environment. The present inventionalso envisages that the photocatalytic unit of the present inventioncomprises an activity greater than that of wild type photocatalytic unitin its in-vivo environment.

In order for the modified photocatalytic units of the present inventionto comprise photocatalytic activity following attachment to a solidsupport, the photocatalytic units must be attached in an oriented mannerso that they will not neutralize each others charge. As described inExample 3, a modified polypeptide of the present invention enablesphotocatalytic unit binding to a solid support such that a light-inducedpositive potential developed. The induced potential is a result of anegative charge displacement away from the gold side of PS I. Thepresent inventors hypothesized that by substituting an identical aminoacid for cysteine in a plurality of photocatalytic units, the attachmentto a solid support will be oriented and the photocatalytic units wouldform a monolayer on the solid support.

The precise orientation of the photocatalytic units on the solid supportmay be adjusted by selecting a particular amino acid to be substitutedby the cysteine residue.

Methods of measuring photocatalytic activity on surfaces fabricatedtherewith include measuring the photovoltage properties of thefabricated surfaces. The photovoltage properties may be measured forexample by Kelvin probe force microscopy (KPFM). As illustrated in theKPFM images presented in FIGS. 3A and 3B, the photocatalytic units ofthe present invention demonstrate a clear light-induced electricpotential. Specifically, a light-induced positive potential of+0.498±0.02 V developed where peaks ascribed to PS I complexes wereobserved in the topographic trace. The reversible nature of thelight-induced electric potential was also demonstrated by observing achange in the potential when the illumination was turned off (FIGS.4A-B).

Photocatalytic activity may also be measured by analyzing the electrontransfer in the photocatalytic complexes. Electron transfer may bemeasured by analyzing flash-induced absorption changes as measured bysingle turnover spectroscopy. As illustrated in FIGS. 6A-B, thedifference in the rate of charge recombination between that of the wildtype and the mutant indicates that the cysteine substitution accordingto the teachings of the present invention did not alter the mode ofaction of light-induced electron transfer.

Reference is now made to FIG. 7, which is a schematic illustration of anoptoelectronic device 10, according to various exemplary embodiments ofthe present invention. Device 10 comprises a solid support 12 and aplurality of isolated photocatalytic units 14 attached to a surface 13of support 12. Isolated photocatalytic units 14 are preferably modifiedso as to facilitate covalent attachment of units 14 to surface 13, whilemaintaining the photocatalytic activity as further detailed hereinabove.

Being compose in part of photocatalytic units 14, optoelectronic device10 facilitates light induced electron transfer. Upon excitation by light11, an electron transfer occurs from a donor site 16, across multipleintermediate steps to an acceptor site 18, within a period of time whichcan be from several hundreds of picoseconds to a few microseconds,depending on the type of photocatalytic units. The frequency of lightwhich induces the electron transfer depends on the photosyntheticorganisms from which units 14 are obtained. For example, whenphotocatalytic units of green plants or green bacteria are employed,device 10 is sensitive to green light having wavelength of from about400 nm to about 750 nm, when photocatalytic units of cyanobacteria areemployed, device 10 is sensitive to cyan light having wavelength of fromabout 400 nm to about 500 nm, when photocatalytic units of red algae areemployed, device 10 is sensitive to red light having wavelength of fromabout 650 nm to about 700 nm and when photocatalytic units of purplebacteria are employed, device 10 is sensitive to purple light havingwavelength of from about 400 nm to about 850 nm.

Optoelectronic device 10 can be used in the field of micro- andsub-microelectronic circuitry and devices including, but not limited tospatial imaging devices, solar batteries, optical computing and logicgates, optoelectronic switches, diodes, photonic A/D converters, andthin film “flexible” photovoltaic structures.

Reference is now made to FIG. 8, which is a schematic illustration of aphotodiode device 20, according to various exemplary embodiments of thepresent invention. One skilled in the art will recognize that severalcomponents appearing in FIG. 7 have been omitted from FIG. 8 for clarityof presentation. Photodiode device 20 comprises optoelectric device 10,and two electrical contacts 22 and 24 being in electrical communicationwith donor site 16 and acceptor site 18, respectively. Electricalcommunication with donor site 16 can be established, for example, byconnecting a conducting material to support 12 or surface 13. Theacceptor site can be covalently bound by formation of sulfide bondbetween the modified polypeptides of the present invention (e.g. W31C inPsaC subunit of PS I) and the top deposited metal electrode. Platinizedphotocatalytic units at the acceptor side can make a metal to metalelectrical connection with a top electrode deposited by evaporation ofthin metal electrode. Deposition of conducting polymer on top of thephotocatalytic monolayer or the platinized photocatalytic monolayer canserve as a top electrode. A symbolic illustration of the photodiode isillustrated at the bottom of FIG. 8.

In use, the photocatalytic units are irradiated by light hence beingexcited to efficient charge separation of high quantum efficiency, whichis typically above 95%. Contacts 22 and 24 tap off the electricalcurrent caused by the charge separation. Depending on the voltageapplied between contacts 22 and 24, photodiode device 20 can be usedeither as a photovoltaic device, or as a reversed bias photodiode.

Specifically, in the absence of external voltage, photodiode device 20enacts a photovoltaic device which produces current when irradiated bylight. Such device can serve as a component in, e.g., a solar cell.

When reverse bias is applied between contacts 22 and 24, photodiodedevice 20 maintains high resistance to electric current flowing fromcontact 24 to contact 22 as long as photodiode device 20 is notirradiated by light which excites the photocatalytic units. Uponirradiation by light at the appropriate wavelength, the resistance issignificantly reduced. Such device can serve as a component in, e.g., alight detector.

Optoelectronic device 10 can also serve as a solar cell, when no biasvoltage is applied. Upon irradiation of the photocatalytic units, thecharge-separated state results in internal voltage between donor site 16and acceptor site 18. The internal voltage can be tapped off viaelectrical contacts at donor site 16 and acceptor site 18. If thecurrent circuit is closed externally, the current flow is maintainedthrough repeated light-driven charge separation in the solar cell.

The generated polarized charge-separated state of device 10 can also beutilized for in a molecular transistor. Specifically, device 10 canserve as a light-charged capacitor enacting a gate electrode whichmodifies the density of charge carriers in a channel connected thereto.

Reference is now made to FIG. 9, which is a schematic illustration of aphototransistor 30, according to various exemplary embodiments of thepresent invention. Phototransistor 30 comprises a source electrode 32, adrain electrode 34, a channel 36 and a light responsive gate electrode38. Gate electrode 38 preferably comprises optoelectronic device 10.Channel 36 preferably has semiconducting properties such that thedensity of charge carriers can be varied.

In the absence of light, channel 36 does not contain any free chargecarriers and is essentially an insulator. Upon exposure to light, thephotocatalytic units of device 10 generate a polarized charge-separatedstate and the electric field caused thereby attracts electrons (or moregenerally, charge carriers) from source electrode 32 and drain electrode34, so that channel 36 becomes conducting. Thus, phototransistor 30serves as an amplifier or a switching device where the light controlsthe current flowing from source electrode 32 and drain electrode 34.

The electrodes can be made of any conducting material, such as, but notlimited to, gold. The inter-electrode spacing determines the channellength. The electrodes can be deposited on a semiconductor surface toform the source-channel-drain structure. The gate electrode can beformed from the isolated photocatalytic units of the present embodimentsas further detailed hereinabove. A symbolic illustration of thephototransistor is illustrated at the right had side of FIG. 9.

As will be appreciated by one ordinarily skilled in the art,phototransistor 30 can operate while gate electrode 38 is left an opencircuit because the gating is induced by photons impinging on electrode38. Phototransistor 30 can be used as a logical element whereby thephototransistor can be switched to an “on” state by the incident light.In addition, phototransistor 30 can be used as the backbone of an imagesensor with large patterning possible due to a strong variation of thedrain current with the spatial position of the incident light beam.Several phototransistors, each operating at a different wavelength asfurther detailed hereinabove can be assembled to allow sensitivity ofthe image sensor to color images. The charge storage capability of thestructure with further modifications known to one skilled in the art ofconventional semiconductors can be exploited for memory relatedapplications.

Photodiode 20 and/or phototransistor 30 can be integrated in manyelectronic circuitries. In particular, such devices can be used asbuilding blocks which can be assembled on a surface structure to form acomposite electronic assembly. For example, two or more photodiodes orphototransistors can be assembled on a surface structure to form a logicgate, a combination of logic gates or a microprocessor.

Reference is now made to FIG. 10 which is a simplified illustration ofan optocoupler 40, according to various exemplary embodiments of thepresent invention. Optocoupler 40 is particularly useful fortransferring signals from one element to another without establishing adirect electrical contact between the elements, e.g., due to voltagelevel mismatch. For example, optocoupler 40 can be used to establishcontact free communication between a microprocessor operating at lowvoltage level and a gated switching device operating at high voltagelevel.

According to a preferred embodiment of the present invention optocoupler40 comprises an optical transmitter 42 and an optical receiver 44.Transmitter 42 can be any light source, such as, but not limited to, alight emitting diode (LED). Receiver 44 preferably comprisesoptoelectronic device 10, and can be, for example, a photodiode (e.g.,photodiode 20) or a phototransistor (e.g., phototransistor 30).Transmitter 42 is selected such that the radiation emitted thereby is atsufficient energy to induce charge separation between donor site 16 andacceptor site 18 of device 10.

Transmitter 42 and receiver 44 are kept at optical communication butelectrically decoupled. For example, transmitter 42 and receiver 44 canbe separated by a transparent barrier 46 which allows the passage oflight but prevents any electrical current flow thereacross. Transmitter42 and receiver 44 preferably oppose each other such that the radiationemitted from transmitter 42 strikes receiver 44.

Triggered by an electrical signal, transmitter 42 emits light 48 whichpasses through barrier 46 and strikes receiver 44. In turn, receiver 44generates an electrical signal which can be tapped off via suitableelectrical contacts as further detailed hereinabove. Thus optocoupler 40successfully transmits to its output (receiver 44) an electrical signalapplied at its input (transmitter 42), devoid of any electrical contactbetween the input and the output.

Reference is now made to FIGS. 11 a-b, which are simplifiedillustrations of an optoelectronic device 50, according to variousexemplary embodiments of the present invention.

In its simplest configuration, device 50 comprises one or more layers 52of photoactive nanoparticles 54. Nanoparticles 54 are interposed betweentwo electrodes 56 and 57. In the representative example shown in FIG.11, electrode 57 is light transmissive. Electrode 56 can be lighttransmissive, light reflective or light absorptive.

As used herein, “a photoactive nanoparticle” refers to a particle whichchanges its electric dipole when irradiated by light. A photoactivenanoparticle can be, for example, a non-polarized nanoparticle whichbecomes electrically polarized when irradiated by light, or ananoparticle characterized by a certain charge separation and which, inresponse to light, changes (typically increases) its charge separation.

In various exemplary embodiments of the invention nanoparticles 54comprise photocatalytic units of a photosynthetic organism. For example,nanoparticles 54 can comprise surface 13 covalently attached tophotocatalytic units 14, e.g., PS I, as further detailed hereinabove.

In use, electrode 57 is irradiated by light 11 which penetrateselectrode 57 to impinge on layers 52. Each photoactive nanoparticleabsorbs the energy of the light resulting in an electric dipole directedfrom electrode 56 to electrode 57 or vice versa. A potential differenceis thus generated between electrodes 56 and 57. Electrical currentcaused by the potential difference can then be tapped off by electricalcontacts as further detailed hereinabove. Thus, layers 56 and 57 serveas electron and hole injection contacts and device 50 generates aphotocurrent in response to light.

In various exemplary embodiments of the invention the work functions ofelectrodes 56 and 57 differ. Preferably, the work function of electrode56 is lower than the work function of electrode 57. The work function ofa substance is defined as the minimal energy required for removing anelectron from the substance into the vacuum. According to a preferredembodiment of the present invention, layer 56 is a low work functionelectrode.

As used herein, the term “low work-function” refers to a work-functionof 4.5 eV or less, more preferably 4 eV or less.

Suitable low work function materials include, without limitation,alkaline metals, Group 2A, or alkaline earth metals, and Group IIImetals including rare earth metals and the actinide group metals. Alsocontemplated are the Group IB metals, metals in Groups IV, V and VI andthe Group VIII transition metals. More specific examples of low workfunction materials, include, without limitation, lithium, magnesium,calcium, aluminum, indium, copper, silver, tin, lead, bismuth, telluriumand antimony.

According to a preferred embodiment of the present invention aluminum,layer 57 is a high work function electrode.

As used herein, the term “high work-function” refers to a work-functionof 4.5 eV or more, more preferably 5 eV or more.

Suitable high work function materials include materials having any oneof InSnO₂, SnO₂ and zinc oxide (ZnO) metal alloys. Other than thesealloys, oxides of Sn and Zn may also be contained in the material ofelectrode 57.

FIG. 12 illustrates an energy-level diagram in the preferred embodimentin which electrode 56 is made of aluminum and electrode 57 is made ofITO. The internal electric field generated between the electrodes issufficiently high to generate electric field that higher than theelectron-cation pair excitonic energy.

According to a preferred embodiment of the present invention device 50comprises a dielectric layer 64 deposited on electrode 56. Dielectriclayer has a cavity 66 which exposes electrode 56. In this embodiment,layer(s) 52 are preferably placed in cavity 66 such that the photoactivenanoparticles contact electrode 56 at the base of the cavity andelectrode 57 at the top of the cavity. Device 50 preferably comprises asubstrate 62 which serves for carrying electrode 56 and layer 64. Two ormore electrical contacts 58 are preferably attached to or formed onsubstrate 62. Contacts 58 are in electrical communication withelectrodes 56 and 57 so as to tap off the electrical current of device50.

In various exemplary embodiments of the invention the sizes of the aboveelectronic devices (including, without limitation, the optoelectronicdevice, solar cell, photodiode, phototransistor, logic gate andoptocoupler) are in the sub millimeter range. Preferably, the size ofthe electronic devices is from about 0.1 nm to about 100 μm, morepreferably, from about 0.1 nm to about 1 μm.

Reference is now made to FIGS. 13 a-b, which are schematic illustrationsof an optoelectronic array 60, according to various exemplaryembodiments of the present invention. Optoelectronic array 60 comprisesseveral optoelectronic devices similar to device 50 arranged array-wiseon a substrate 62, for example, a silicon substrate or the like. Theadvantage of using an optoelectronic array is that such configurationcan facilitates up-scaling of the physical dimensions of theoptoelectronic device to amplify the photovoltaic signal. It was foundby the Inventors of the present invention that the dimension of suchoptoelectronic array can be from several microns to a few centimeters.

The electric configuration between the optoelectronic devices of array60 depends on the desired output. For current output, the preferredelectric configuration is serial, whereas for voltage output a parallelconfiguration is more preferred. The arrangement of the optoelectronicdevices on substrate 62 is preferably such that several optoelectronicdevices share the same electrodes. This can be achieved in anygeometrical arrangement. For example, referring to FIG. 13 b, twoconductive layers and a dielectric layer separating one layer from theother can be deposited on substrate 62. One conductive layer can includeelectrodes of the type of, e.g., electrode 56, and another conductivelayer can include electrodes of the type of, e.g., electrode 57. Theelectrodes of the conductive layers are preferably arranged inorthogonal or any other no-parallel directions. The photoactiveparticles of device 50 are introduced into cavities formed in thedielectric layer at the intersections between the electrodes of onelayer and the electrodes of the other layer, such that each suchintersection defines one optoelectronic device. A preferred process forfabricating array 60 is provided hereinunder with reference to FIG. 16a-d.

Reference is now made to FIGS. 14 and 15 a-d which are a flowchartdiagram (FIG. 14) and schematic process illustrations (FIGS. 15 a-d) ofa method suitable for fabricating an optoelectronic device, according tovarious exemplary embodiments of the present invention.

It is to be understood that, unless otherwise defined, the method stepsdescribed hereinbelow can be executed either contemporaneously orsequentially in many combinations or orders of execution. Additionally,one or more method steps described below are optional and may not beexecuted.

The method begins at step 70 and optionally and preferably continues tostep 71 in which a first electrode is deposited on a substrate. FIG. 15a illustrate first electrode 56 deposited on a substrate 62. The firstelectrode, as stated, is preferably an electron-injection electrodewhich can be light transmissive, light reflective or light absorptive asdesired. Step 71 can be executed by evaporation followed byphotolithography and etching. For example, gold metal can be evaporatedon a substrate silicon dioxide layer. The gold layer can then patternedby photolithography according to the desired shape of the firstelectrode. Subsequently, the electrode can be shaped by etching.

The method continues to step 72 in which photocatalytic units arecovalently attached to the first electrode to provide a first layer ofphotoactive nanoparticles as further detailed hereinabove. The top sideof the photoactive nanoparticles preferably comprises a conductingmoiety to allow attachment of other photoactive nanoparticles.

The method then continues to step 73 in which one or more layers of thephotoactive nanoparticles are attached to the first layer electrode (seeFIG. 15 b), to provide a plurality of layers of photoactivenanoparticles. Step 73 can be repeated one or more time, depending onthe number of photoactive nanoparticle layers of the device.

Step 72 preferably comprises fabrication of a cavity 66, e.g., byforming a cavity through a dielectric layer 64 on top of first electrode56 and substrate 62. The dielectric layer can be made of any dielectricmaterial suitable for the process by which the cavity is formed. Forexample, a layer of silicon nitride can be deposited on top of the firstelectrode, e.g., using Chemical Vapor Deposition (CVD), physical vapordeposition (PVD), or atomic layer deposition (ALD). The cavity can thenbe formed in the dielectric layer (silicon nitride, in the presentexample) by photolithography followed by etching. In any event, cavity66 is formed such that first electrode 56 is exposed on the base of thecavity, to allow adsorption of the photoactive nanoparticles on thefirst electrode.

The preferred adsorption technique depends on the type of photoactivenanoparticles. In various exemplary embodiments of the invention lightinduced adsorption is employed. When the nanoparticles comprisephotocatalytic units having a modified polypeptide, the nanoparticlesattach to the first electrode via the amino acids at the modified site.For example, thiolated PS I nanoparticles can be attached via theirthiol moiety to form a stable oriented self assembled monolayer (SAM).Light induced adsorption can be used to adsorb the PS I nanoparticlesinto a dense layer.

Chemical bonding to the second electrode of the device can be improvedby photoreducing Pt⁴⁺ ions in solution by PS I monolayer. Such aprocedure was earlier used for platinization of PS I in suspension[Millsaps, J. F.; Bruce, B. D.; Lee, J. W.; Greenbaum, E. Photochemistryand Photobiology 2001, 73, 630-635]. The procedure results in localdeposition of Pt at the electron donor end of the protein. A freshincubation of the platinized monolayer with cycteine mutants of PS Iresults in formation of sulfide bond between the cystiene in the PS Iand the platized top of the monolayer to form a second oriented SAM.These cycles are preferably repeated so as to form of an orientedmultilayer inside the cavity (see FIG. 15 c).

The method continues to step 74 in which the second electrode isdeposited on the layer(s) of photoactive nanoparticles (see FIG. 15 d).The second electrode, as stated, is preferably a hole-injection lighttransmissive electrode and it can be any electrode as long as it iscapable of functioning as an anode so as to inject holes into the layersof nanoparticles. Preferably, the second electrode comprises ITO whichcan be deposited by sputtering, electron beam vapor deposition, ionplating, indirect evaporation process etc. In various exemplaryembodiments of the invention ITO clusters are deposited on thenanoparticles with relatively very low momentum and temperature, so asto prevent or minimize the destruction of the nanoparticles.

The method ends at step 75.

Reference is now made to FIGS. 16 a-d which are schematic illustrationsof a preferred process for an optoelectronic array, according to variousexemplary embodiments of the present invention. With reference to FIG.16 a, a plurality of electrodes of the type of, e.g., electrode 56, isdeposited on substrate 62. The technique for depositing the electrodescan be similar to the technique described above. For example, aconductive layer can be evaporated on the substrate and,photolithography followed by etching can be employed to form theelectrodes on the evaporated layer. In the simplified illustration shownin FIG. 16 a, electrodes 56 are conveniently shaped as a plurality ofparallel stripes, but it is not intended to exclude any other shape forthe electrodes.

With reference to FIGS. 16 b-c, dielectric layer 64 is deposited on topof electrodes 56 and a plurality of cavities 66 are formed in dielectriclayer 64 by photolithography followed by etching to expose electrode 56as further detailed hereinabove.

Once the cavities are formed, the nanoparticles can be introduced intothe cavities as further detailed hereinabove. A plurality of electrodesof the type of, e.g., electrode 57, is then deposited on layer 64 so asto contact the nanoparticle in cavities 66. Electrodes 57 areillustrated in FIG. 16 d as a plurality of parallel stripes,substantially orthogonal to electrodes 56. Other shapes for electrodes57 are also contemplated, provided the nanoparticles in the cavitiesinterconnect electrodes 56 and 57.

Additional objects, advantages and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Synthesis of Synechocystis sp. PCC 6803 psaB Mutants

The robust PS I reaction center from the cyanobacteria Synechosystis sp.PCC 6803 was selected to ascertain whether genetic modifications couldassist in attachment of the reaction center to a solid support. The mainreason for the structural stability of this PS I is due to the fact thatall chlorophyll molecules and carotenoids are integrated into the coresubunits complex while in plant and other bacterial reaction centers theantenna chlorophylls are bound to chlorophyll-protein complexes that areattached to the core subunits.

Selection of the amino acids to be modified to cysteines for covalentattachment of the PS I to the gold surface was based on the knowledge ofthe atomic structure of the PS I. Thus, amino acids in the extramembrane loops facing the cytoplasmic side of the bacterial membranewhich do not have stereo hindrance when placed on a solid surface weremutated to cysteines in order to ensure formation of sulfide bonds. Thevarious mutations were selected near the P700 to secure close proximitybetween the reaction center and the gold electrode in order tofacilitate efficient electric junction.

Methods and Materials

Introduction of mutations in psaB of Synechocystis sp. PCC 6803 byhomologous recombination: Site-directed mutagenesis in the psaB gene waseffected by homologous recombination. A 1.8 kb psaB gene fragment and a1.1 kb downstream flanking region were inserted into the pBluescript IIKS vector (see scheme in FIGS. 1A-C). A 1.27 kb kanamycin resistanceconferring gene (Kan^(R)) was excised from pUC4K and inserted at theEcoRI site at the beginning of the flanking to construct vector pZBL aspreviously described [Zeng et al., Biochim. Biophys. Acta 2002, 1556254-264]. An overlapping extension PCR [Ho et al., Gene 1989, 77 51-59]was used for construction of an extended fragment 1400 bp, containingthe mutations, and vectors pZBL D480C, pZL S500C, pZBL-S600C, pZBLY635C, and pZBL D236C and pZBL-S247C, were constructed by fragmentexchange at restriction sites Sph I-Hpa I and Sph I-Sac I, respectively.

The primers used to generate the polypeptides of the present inventionare presented herein below in Table 4.

TABLE 4 SEQ ID Muta- Plas- NO: Sequences (5′→3′) tions mids psaB SEQ IDGTGTATGCGGCGTGTCCCGACACTGCTGGC D235C PZBL- NO: 1 D235C SEQ IDAGCAGTGTCGGGACACGCCGCATACACGCC D235C pZBL- NO: 2 D235C SEQ IDCACATTTTTGGTTGTTCTGAAGGTGCTGGT S246C pZBL- NO: 3 S246C SEQ IDAGCACCTTCAGAACAACCAAAAATGTGGCC S246C pZBL- NO: 4 S246C SEQ IDCTCTCCAATCCTTGCAGCATTGCTTCCACC D479C pZBL- NO: 5 D479C SEQ IDGGAAGCAATGCTGCAAGGATTGGAGAGCAA D479C pZBL- NO: 6 D479C SEQ IDGATGCTATCAACTGCGGCACCAACTCTCTG S499C pBL- NO: 7 S499C SEQ IDAGAGTTGGTGCCGCAGTTGATAGCATCCAA S499C pZBL- NO: 8 S499C SEQ IDCTCGGTGTTTGGTGCGGTAACGTTGCTCAG S599G pZBL- NO: 9 S599C SEQ IDAGCAACGTTACCGCACCAAACACCGAGGTG S599C pZBL- NO: 10 S599C SEQ IDGGTTACAACCCCTGCGGTGTCAACAATCTG Y634C pZBL- NO: 11 Y634C SEQ IDATTGTTGACACCGCAGGGGTTGTAACCATT Y634C pZBL- NO: 12 Y634C SEQ IDGTGTATGCGGCGTGTCCCGACACTGCTGGC D235C/ pZBL- NO: 13GGTTACAACCCCTGCGGTGTCAACAATCTG Y634C D235C/ Y634C SEQ IDAGCAGTGTCGGGACACGCCGCATACACGCC D235C/ pZBL- NO: 14ATTGTTGACACCGCAGGGGTTGTAACCATT Y634C D235C/ Y634C SEQ IDCACATTTTTGGTTGTTCTGAAGGTGCTGGT S246C/ pZBL- NO: 15GGTTACAACCCCTGCGGTGTCAACAATCTG Y634C S246C/ Y634C SEQ IDAGCACCTTCAGAACAACCAAAAATGTGGCC S246C/ pZBL- NO: 16ATTGTTGACACCGCAGGGGTTGTAACCATT Y634C S246C/ Y634C psa C SEQ IDGAAATGGTGCCCTGGTGTGGTTGTAAAGCC F31C P61- NO: 17 2.4- F31C SEQ IDAGCGGCTTTACAACCACACCAGGGCACCAT RW31C P61- NO: 18 2.4- R3LC SEQ IDAGATCTTTAGTGGTGGTGGTGGTGGTGGTAA His- P61- NO: 19          GCTAAACCCATtaq C- 2.4- His- term His- taq C- taq C- term. term Table 4 continued

In addition, a single mutation at W31C was also inserted in PsaC and twodouble mutations (D236C/Y635C and S247C/Y635C) were inserted in PsaBusing the same techniques described above.

For selection of psaB deficient recipient cells, a pBLΔB vector wasconstructed by excision of 1.3 kb from the downstream end of psaB (fromSphI to EcoRI from pZBL) and insertion of a 1.3 kb Chloramphenicolresistant confering gene (Cm^(R)) at these sites (FIG. 1C). Wild typeSynechocystis cells were transformed with pPLΔB, and the transformentsgrown under “light adapted heterotrophic” conditions [Zeng et al.,Biochim. Biophys. Acta 2002, 1556 254-264] to express the D480C mutantpolypeptide (SEQ ID NO: 20), S500C mutant polypeptide (SEQ ID NO: 21),S600C mutant polypeptide (SEQ ID NO: 22), Y635C mutant polypeptide (SEQID NO: 23), D236C mutant polypeptide (SEQ ID NO: 24), S247C mutantpolypeptide (SEQ ID NO: 25) and W31C mutant polypeptide (SEQ ID NO: 26).

Isolation of thylakoid membranes and PS I complexes: The Synechocystiscells were broken in a French pressure cell at 500 p.s.i. and thylakoidswere isolated by differential centrifugation. PS I was solublilized bythe detergent n-dodecyl β-D-maltoside and purified on DEAE-cellulosecolumns and on a sucrose gradient [Nechushtai, R., Muster, P., Binder,A., Liveanu, V., and Nelson, N. (1983) Proc. Natl. Acad. Sci. USA 80,1179-1183]. The analysis of subunit composition by SDS polyacrylamidegel electrophoresis and Western blotting were performed as previouslydescribed [Laemmli, U. K. (1970) Nature 227, 680-685; Tindall, K. R. andKunkel, T. A. (1988) Biochemistry 27, 6008-6013]. Protein in themembranes was determined after solubilization in 1% SDS as described[Lowry, O. H., Rosenbrough, N. L., Farr, A. L., and Randall, R. J.(1951) J. Biol. Chem. 193, 265-275]. Chlorophyll concentration and P700chemical- and photo-oxidation were determined according to a publishedmethod [Arnon, D. I. (1949) Plant Physiol. 24, 1-15]. The detailedisolation procedure and analysis are summarized in [Gong et al., Journalof Biological Chemistry 2003, 278 19141-19150]. The analysis confirmedthe isolation product is purified protein chlorophyll complex of PS I.

Surface-exposed cysteines on PS I were probed by biotin-maleimide whichspecifically reacts with the sulfhydryl groups. Biotin-labeled PS Icomplexes were dissociated and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. For immunoblot detection, proteinsamples were transferred from the gel to nitrocellulose and reacted withperoxidase-conjugated avidin, then developed with enhancedchemiluminescence reagents as previously described [Sun et al., Methodsin Enzymology, Academic Press, 1998, p. pp. 124-139].

Results

As illustrated in FIG. 1E, all modified purifed PS I units comprisedsurface-exposed cysteines. Although, the non-modified protein contains 9free cysteines none of them were found to be exposed to the externalsurface when tested with the surface active reagent biotin-maleimide.

Example 2 Fabrication of Oriented Monolayers

Methods and Materials

Fabrication: The fabrication of orientated monolayers was carried out bydirectly reacting the cysteine in the mutant PS I with a fresh, clean,hydrophilic gold surface to form an Au-sulfide bond. A flat gold surfacewas prepared by evaporation of 5 nm of Cr and 150 nm of gold on a glassor silicon wafers. These surfaces were annealed at 350° C. for 1 h undervacuum. A buffered solution containing 1 mg chlorophyll of PS I in 1 mlwas layered on top of the metal surface for incubation. Following a twohour incubation of either mutant or native PS I at room temperature, theunattached proteins were thoroughly washed with distilled water severaltimes. The surface was dried with ultrapure nitrogen and observed usingatomic force microscopy (AFM).

Atomic Force Microscopy (AFM): All measurements were made with acommercial AFM (Nanoscope® IIIa MultyMode™ with Extender™ ElectronicsModule, Veeco Instruments). The topography measurements were conductedin tapping mode at a cantilever resonance frequency of 300 kHz.

Results

PS I mutants self-assembled on the gold surfaces following annealing andremained covalently attached to the gold surface following the two hourincubation. Native PS I which were incubated in a similar manner werewashed away from the gold surface. FIG. 2B illustrates atomic forcemicroscopy images obtained by a scan of a 0.3 μm² area of the goldsurface on the glass wafer, to which a monomer, of PS I cysteine mutantD480C was attached through sulfide bonds. FIG. 2B clearly shows amonolayer of particles 15-21 nm in diameter and 9 nm high. This is theexpected size of PS I as obtained by crystallography. These particlesare seen over the lightly granulated surface. FIG. 2A illustrates a scanof the annealed untreated naked gold surface, the annealed gold granulesare 150 nm in diameter and 5 nm high.

Example 3 Photovoltage Properties of the PS I Mutant Fabricated Surfaces

The photovoltage of single PS I trimer and monomer complexes of cysteinemutant D480C in the monolayer was measured by Kelvin probe forcemicroscopy (KPFM).

Materials and Methods

The KPFM setup is based on a commercial AFM (modified NanoScope® IIIaMultiMode, Veeco, USA) operating in tapping mode. The electrostaticforce is measured in the so-called ‘lift mode’. Essentially, after thetopography is measured the tip is retracted from the sample surface to afixed height. The oscillation of the tip induced by the piezo is stoppedand an AC bias is applied to the cantilever at the same frequency usedbefore for the topography measurements in the tapping mode. The CPD isextracted in the conventional way by nullifying the output signal of alock-in amplifier which measures the electrostatic force at the firstresonance frequency [Vatel et al., Journal of Applied Physics 1995,772358-2362]. AFM topography and the corresponding KPFM electricpotential were recorded in sequential scans at a scan rate of 1 Hz; 512lines were scanned in two segments over the sample area to formtwo-dimensional image. A helium-neon laser (λ=632.8 nm, 5 mW/cm²) wasswitched on for the first segment of the scan and off for the second.

Results

As illustrated in FIGS. 3A and 3B, the KPFM images demonstrate a clearlight-induced electric potential in mutant PS I complexes. Alight-induced positive potential of +0.498±0.02 V (see Table 1hereinbelow) developed where peaks ascribed to PS I complexes wereobserved in the topographic trace (FIGS. 3A and 3B).

These results clearly indicate that all mutant PS I complexes bound tothe gold surface were functionally active and oriented in the samedirection. The induced potential is a result of a negative chargedisplacement away from the gold side of PS I as shown in FIG. 1D.

The measured change in contact potential difference (CPD) underillumination is in a good agreement with a simple calculation of thepotential change (ΔΦ) resulting from an induced dipole layer of a heightd, at an angle θ with respect to the surface normal given by: Δφ=Nqd cosθ/ε, where N is the area density of the dipole layer, q is theelementary charge, and ε is the layer dielectric constant. Using N of2.2×10¹¹ cm⁻² for the trimmer, d=5 nm, ε=2, and θ=90⁰ we obtain ΔΦ=0.41Volt. The discrepancy between the expected +1 V and the calculated 0.41V is partially resolved because the angle of the dipoles in the layer isnot known, and also the dielectric constant is an ill-defined propertysince the dipole layer is not a bulk entity. Moreover, as the reactioncenters were relatively far from each other the measured CPD underillumination was much less then the real CPD. Due to the long-rangeelectrostatic forces, the measured CPD at a point on the surface belowthe tip apex is a weighted average of the surface potential in thevicinity of the tip. The effect of the tip electrostatic averaging hasbeen calculated in the past using different algorithms [Y. Rosenwaks etal., Physical Review B 2004, 70 085320-085327]. Based on thesecalculations it is estimated that a more accurate quantity of the CPD(under illumination) is around a factor of two larger then the measuredone. A similar light induced potential was measured in plant PS I placedon mercaptoethanol coated gold [R. Lee et al., J. Phys. Chem. B 2000,104 2439-2443]. In these experiments, The PS I was insulated from thegold by the mercaptoethanol in a loosely bound monolayer and only 70% ofthe complexes assumed the same orientation. The gold surface workfunction also increased by approximately +0.125 V during illumination.Partial charge transfer to the vicinity of the photo-oxidized P700 whichis located at the interface of PS I and the metal is expected to resultin a more negative substrate. The charge transfer is indicative ofefficient electronic coupling between the gold and PS I. There washowever, very little change in the CPD when untreated gold surface wasilluminated. It can also be seen that the PS I complexes had a slightlymore positive potential (CPD of +0.2 V) than the gold substrate in thedark (FIG. 3B). This potential might have resulted from excitation of PSI by the AFM feedback laser that was not completely masked by thecantilever used in the KPFM setup. Such background excitation mightcause an under estimation of the light minus dark CPD change in PS I.

The reversible nature of the light-induced electric potential wasdemonstrated by observing a change in the potential when theillumination was turned off. The results are provided in Table 5 hereinbelow. The values represent an average potential of 24 individual PS Icomplexes measured either in the light or in the dark.

TABLE 5 PS I/Potential Dark (V) Light (V) Light to dark (V) Dark tolight (V) monomer −0.193 ± 0.0005 +0.118 ± 0.0010 +0.311 ± 0.0010 —monomer −0.353 ± 0.0002 +0.004 ± 0.0006 — +0.356 ± 0.001 trimer +0.870 ±0.003  +1.220 ± 0.0200 +0.358 ± 0.0014 — trimer +0.717 ± 0.003  +1.215 ±0.0200 — +0.498 ± 0.020 The measurements were started either in thelight and then light was turned off (light to dark) or started in darkand then illumination was turned on (dark to light).

As detailed in Table 5, the illuminated trimers of PS I cysteine mutantsin the monolayer developed a CPD of +1.220±0.02 V that decreased to+0.870±0.003 V when light was turned off (see FIG. 4B).

The substrate potential did not change when the light was turned off.Under the applied bias potential in the experiments charges could notmove quickly to the balk gold substrate from the vicinity of PS I.Therefore, the light minus dark CPD (SPV) was smaller when the light wasturned off compared to the difference on turning light on (Table 5). Theaverage light minus dark potential difference of multiple PS I complexeswas +0.358±0.014 V. There was no change in the contact potential wherethe gold surface was exposed. The photopotential could be reproducedmultiple times repeatedly on the same sample or on a sample that wasstored for over a month.

The PS I monomers of the cysteine mutant D480C readily formedself-assembled oriented monolayers. The average distance between themonomers (FIG. 2B) and the trimers (FIG. 5B) in the monolayers was 15 nmand 25 nm, respectively. The small monomers were more densely packedthan the trimers in the monolayer yet, they could be resolved by the AFMmeasurements with the high resolution cantilever tip (FIG. 2B). However,neither the topography nor the CPD measurements were sensitive enough toresolve the individual monomers. Therefore, the CPD obtained by KPFMmeasurements in the dark of the PS I monomer monolayer describes acontinuum (FIG. 5A). Similar images were observed when the topographywas determined by the same setup (not shown). Yet, following lightillumination, the CPD increased. An average of measurements of CPD atmultiple locations on the monolayer in dark and in the light gavelight-induced CPD of +0.356±0.001V (Table 5). A smaller light-inducedCPD of +0.311±0.001 V was observed when the change was determined onturning off the illuminated monolayer.

Example 4 Self Assembly of other Cysteine Mutants of the PresentInvention

Self assembly of oriented PS I was also obtained with three othermutants in which amino acids S500C, S600C, Y635C located at the exposedextra-membrane loops were modified to cysteines (see FIG. 1D). Bothmonomers and trimers of PS I of these mutants formed monolayers thatgenerated light-induced CPD of similar magnitude to the one measured inmonolayers fabricated by mutant D480C. Therefore, a functional orientedmonolayer of PS I depends on formation of a sulfide bond between acysteine located at the extra-membrane loop of the complex and is notconfined to a specific location.

Example 5 Kinetic Analysis of Charge Recombination in PS I

To characterize the effects of the mutations on the electron transfer inthe PS I complexes, flash-induced absorption changes were measured bysingle turnover spectroscopy.

Materials and Methods

Spectroscopic measurements: Measurements of P700 photooxidation at 700nm and at 820 nm in thylakoids and PS I used a modified flash photolysissetup as earlier described described [Gong et al., Journal of BiologicalChemistry 2003, 278 19141-19150]. The samples contained 25 mM Tris, pH8, 10 mM sodium ascorbate, 10 μM phenazine methosulfate and 60 μgchlorophyll/ml PS I complexes. Absorption change transients wereanalyzed by fitting with a multiexponential decay using Marquardtleast-squares algorithm programs (KaleidaGraph 3.5 from SynergySoftware, Reading, Pa.).

Results

Light-induced oxidation of P700 causes a decrease in absorption at 700nm or an increase in absorption at 820 nm. The flash-induced transientΔA820 (and at ΔA700 nm, not shown) decay in PS I protein isolated frommutants D480C showed a similar result as in wild type, with a backreaction of 4.5 ms halftime, which may be ascribed to the reduction ofP700⁺ by the electron transfer mediator phenasine mehtosulfate (FIG.6A). Similar results were obtained for S500C and S600C PS I complex. Theresults indicated that electrons are mediated to F_(A)/F_(B) in themutated PS I. If there was a disturbance in the mediation to F_(A)/F_(B)and the reduction of P700⁺ would be a result of reduction from a carrierthat is located prior to F_(A)/F_(B), the rate of recombination would befaster than 4.5 ms. Indeed, mutant S600C absorption decay was resolvedinto two components of 4.5 ms (85%) and 0.5 ms (15%) indicating thatpart of the electron transfer only reached F_(x) resulting in chargerecombination between P700⁺ and F_(X) ⁻ [K. Brettel, Biochim. Biophys.Acta 1997, 1318 322-373]. The similarity of the rate of chargerecombination between that of the wild type and the mutant indicatesthat site-directed mutagenesis does not alter the mode of action oflight-induced electron transfer. These results are in agreement with thefact that the mutants could grow autotrophically in continuous light.

Example 6 Determination of Orientation by X-ray Fluorescence

Materials and Methods

X-ray absorption measurements: X-ray absorption and fluorescence wascollected at undulator beam line Sector 18 ID-D, the BioCAT facility atthe Advanced Photon Source, Argonne National Laboratories, Argonne, Ill.The beam was fed through double Si(III) crystal monochromator whileharmonic rejection was attained by using a harmonic rejection mirror.Focused beam size was 100 μm by 150 μm with flux of 10¹⁴ photons/sec in10⁻⁴ DE/E bandwidth. The incident X-ray beam intensity Io was monitoredby N₂ gas filled ion chamber and X-ray fluorescence was monitored by themultilayer array detector. The Fe K-edge were scanned between X-rayenergies of 7000 eV and 7900 eV. To minimize radiation damage 60 s scanswere taken at each angle on the samples of PS I at 100K.

Results

PS I orientation in self assembled monolayer was determined by totalreflection measurements of grazing x-ray fluorescence. PSI was attachedthrough formation of sulfide bonds between unique cysteine and tungstenon tungsten-carbon multilayer over silicon substrates. Each graph is anaverage of 60, 42 s scans in the indicated angle to the x-ray beamnormal 25, 45, 60 and 90 degrees. As illustrated in FIG. 6B, x-rayfluorescence k-edge changed as a function of the change in the anglerelative to the polarized x-ray beam. Such a change was expected when PSI and the iron-sulfur cluster are oriented relative to the plane of thesilicon substrate. Each of the three [4Fe-4S] iron-sulfur clusters formdistorted cubes that are located at the center and along both sides ofthe pseudo-C₂ symmetry axis of PS I [P. Jordan, et al., Nature 2001, 411909-917]. Therefore, the absorption extinction of a polarized x-ray beamis expected to change as a function of the angle of the PS I pseudosymmetry axis to the polarize beam. Indeed, the orientation of theiron-sulfur clusters was earlier determined in partially orientedthylakoid membranes by electron paramagnetic resonance [R. C. Prince,Biochimica et Biophysica Acta (BBA) Bioenergetics 1980, 592 323-337].

Conclusions

It was demonstrated for the first time in this work that selection of arobust reaction center PS I from cyanobacteria together with a rationaldesign of mutations based on the crystallographic structure enable thefabrication of oriented monolayers on conducting metal surface. Directbinding of the protein complex to the metal electrode through formationof sulfide bond between unique cysteines induced by mutation secured thestability, orientation, function and an efficient electronic junction.The dry membrane protein in the monolayer retained it capacity togenerate photo-potential of approximately +1 V. The photodiodeproperties, the nanometer scale dimension, the high quantum yield andthe almost 60% energy conversion efficiency makes reaction centersintriguing nano-technological devices for applications in molecularelectronics and biotechnology.

Example 7 PS I Based Photoactive Nanoparticles

In accordance with preferred embodiments of the present invention,photoactive PSI nanoparticles were incorporated in a solid statetemplate. Robust PS I reaction centers from the cyanobacteriaSynechosystis sp. PCC 6803 was found to be stable when covalently boundto metal. The main reason for the structural stability of this PS I isdue to the fact that all chlorophyll molecules and carotenoids wereintegrated into the core subunits complex, while in plant and bacterialreaction centers the antenna chlorophylls are bound tochlorophyll-protein complexes that are attached to the core subunits. Nopeptide surfactants were required for stabilization.

The selection of the amino acids that were modified to cysteines forcovalent attachment of the PS I to the gold surface consisted a secondfactor that insured structural and functional stability of theself-assembled oriented PS I. Amino acids in the extra membrane loopsfacing the cytoplasmic side of the bacterial membrane (D480C, S500C,Y635C) that do not have stereo hindrance when placed on a solid surfacewere mutated to cysteines in order to insure formation of sulfide bond.

The mutations did not modify the photochemical properties and thesubunit composition of the isolated PS I. Oriented monolayers werefabricated by directly reacting the cysteine in the mutant PS I with afresh, clean, hydrophilic gold surface to form an Au-sulfide bond.

The orientation and the photoactivity of the monolayers was measured byKelvin probe force microscopy (KPFM) of single PS I trimer complexes ofcysteine mutant in the monolayer.

FIGS. 17 a-b are two-dimensional spatial (FIG. 17 a) and electricpotential (FIG. 17 b) maps of the oriented monolayers. The images are ofthe same set of PS I reaction center trimers from mutant D480C on anAu—Si surface. FIG 17 c shows binding PS I under illumination. Thescanning directions for each raster of the constructed images were fromtop to bottom (from light to dark). The illumination was provided by aHe—Ne laser at 632.8 nm, 5 mW/cm2. The images show a dense monolayer ofparticles 15-21 nm in diameter and 9 nm in height, which is the expectedsize of PS I as obtained by crystallography. Light induced potentialenhanced the affinity to the metal resulting in aformation of a densermonolayer.

The KPFM image demonstrates a clear, light-induced electric potential inPS I. The light-induced positive potential of +1 V was developed wherepeaks ascribed to PS I complexes were observed. These results indicatethat all PS I complexes bound to the gold surface were functionallyactive and oriented in the same direction. The induced potential is aresult of a negative charge displacement away from the gold side of PSI. The reversible nature of the light-induced electric potential wasdemonstrated in an experiment in which a change in the potential wasobserved when the illumination was turned off. The average light minusdark potential difference of multiple PS I complexes was alsoapproximately 1 V. The photopotential was reproduced a plurality oftimes and repeatedly on the same sample or on a sample that was storedfor over a month.

Example 8 Vectorially Oriented Layers of Photoactive Nanoparticles

In accordance with preferred embodiments of the present invention,construct made of vectorially oriented layers of PS I, was prepared. ThePS I layers were electronically connected in a serial fashion. Theprepared construct has many advantages. It can increase the absorptioncross section, increase electronic output and reduce the risk ofshortcut between the top and bottom electrode. It was alreadydemonstrated [Millsaps, supra] that metallic platinum can beprecipitated at the site of electron emergence from the PS I reactioncenter.

The platinization process(PtCl₆)²⁻+4e+hv=Pt↓+6Cl⁻,occurs at pH 7 and room temperature. The source of electrons forreduction is the reducing electrons from the light-activated PSIreaction center itself.

Pt was precipitated on the reducing end of PS I assembled as an orientedmonolayer on gold surface. AFM images of the monolayer show that thesize of the PS I slightly increased as a result of platinization (FIGS.18 a and 18 c). The phase contrast image however shows metal on top ofeach of the PS I (FIGS. 18 b and 18 d). XPS analysis of monolayersindicated 305 Pt atoms per PS I in the platinized monolayer compared tonone in the monolayer of the untreated PS I. The calculation is based onthe finding of a ratio 1/50 Pt/C assuming 16,800 carbon atoms per PS I.

Formation of vectorially oriented multilayers of PS I on a solid goldsurface was performed by sequential binding and platinization of PS I.The initial monolayer was fabricated by formation of sulfide bondsbetween the metal surface and the unique cysteines in the mutant PS I.The washed monolayer was placed in (PtCl₆)²⁻ solution and illuminateduntil the platization of the PS I. The platinized monolayer was washedand incubated again in a solution of cysteine mutants of PS I forbinding by a formation of sulfide bond with the platinum patches on topof the PS I complexes. This process was repeated several times and theformation of new layer of PS I and its platinization was monitored byAFM and changes in the phase contrast. The thickness of the monolayerswas determined by ellipsometry, the Pt content by XIPS and the functionby determination of the photo-potential with KPFM microscopy. Theplatinization enabled vectorially oriented monolayers and goodelectronic coupling between in the serially assembled photosystems.

Electrochemical measurements of photocurrent generated by the PS Imonolayer were done in a three electrode configuration. A workingelectrode, Pt counter electrode, and a Ag/AgCl, 1 M KCl reference. Theworking electrode was illuminated with a 150 W incandescent slidprojector. The potential at the working electrode was set at between−0.4 and 0.04 V versus Ag/AgCl electrode.

The medium contained 0.1 M tris-HCl, pH 7.5 and 0.05 mM metyl viologenefor mediation of electrons between PS I and the electrode. Highphotocurrent of 0.065 mA/Cm² was measured with the PS I monolayer ongold electrode (FIG. 19). Similar results were obtained with theplatized PS I monolayer.

Due to the direct binding of PS I in accordance with the teachings ofthe present embodiments, the obtained photocurrent is 2,160 fold largerthan a photocurrent of 30 nA/Cm² obtained with oriented monolayer ofbacterial reaction center [Trammell, supra].

Example 9 A Vertical Prototype Device

A vertical prototype device was fabricated according to the teaching ofthe present embodiments. The physical dimensions of the prototype devicewere up scaled to amplify the photovoltaic signal. For photovoltaicmeasurements a multi cell array architecture was adopted. Thismultifunction architecture allowed to explore and measure thephotoelectric properties of single cells (the intersection betweenvertical and horizontal lines), and measure of the output photo-voltageand current signals of multiple cells and arrays arranged in series orparallel configurations. This flexibility is achieved by changing theelectrical connections between the pads that are connecting the cellsvia a probe station setup.

The fabrication process began by evaporation of 100-200 nm of gold metalon top of a silicon dioxide layer (silicon wafer). The gold metal servedas the bottom electrode of the device. The shape of the electrode wasdefined by photolithography and produced by wet I/I⁻ etching (FIGS. 20a-c). Subsequently, a dielectric platform layer was formed by depositing50 nm of Si₃N₄ were on top of the gold electrode using CVD. A squarecavity, reaching the gold electrode, was formed in the Si₃N₄ platform byelectron beam lithography followed by etching.

The PSI SAM were then introduced into the formed cavities and ITOelectrodes were deposited by sputtering technique to encapsulate the PSIwithin the cavities. The thus fabricated prototype is illustrated inFIG. 21. The sputtering was by a special technique developed by Prof.David Cahen (Weizmann Institute, Israel). In accordance with thistechnique, ITO clusters were deposited on the SAM with relatively verylow momentum and temperature. Such conditions prevented the destructionof the SAM. The top electrode was then defined by photolithography andwet etching.

FIG. 22 illustrates the set-up used for the photoconductivityexperiments of the prototype device. The measurements of the device wereperformed using Desert-cryogenics probe-station attached to a Keithleylow current source measure unit. A white light source of output powers50 and 100 Watts (giving 1 kW/m² at the sample which is similar to solarirradiance) was used to initiate the photoconductivity process

FIG. 23 shows measurements of the current as a function of the voltage(I/V). As shown the I/V measurements in dark revealed a two back-to backdiode properties. The lack of photovoltage signal can be explained bythe low internal electric field between the contacts (the work functiondifference between ITO and Au is relatively small), and due to theeffects of the interface between the sputtered ITO and the PSI layer.I/V measurements under illumination gave a clear photoconductivityeffect with an average output current of 0.3A/Cm². These resultsdemonstrate photoactivity, low Schottky barrier and good electroniccoupling through the junctions of the two electrodes.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. An isolated polypeptide comprising an amino acid sequence as setforth in SEQ ID NO:
 20. 2. An isolated photocatalytic unit comprisingthe isolated polypeptide of claim
 1. 3. A membrane preparationcomprising the photocatalytic unit of claim
 2. 4. The isolatedphotocatalytic unit of claim 2, being in a monomeric or trimeric form.